Clinical Outcome of Fresh Testicular Spermatozoa and Failure to Retrieve Sperm from Patients with Severe or Non-Obstructive Azo-ospermia of ...
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Research Article
Clinical Outcome of Fresh Testicular Spermatozoa and Failure to
Retrieve Sperm from Patients with Severe or Non-Obstructive Azo-
ospermia of Oocyte Retrieval and the In Vitro Fertilization/Intracyto-
plasmic Sperm Injection Protocol
Mustafa Zakaria1*, Marcuse F Steven2*, Noureddine Louanjli3, Wassym R Senhaji4, Aya Al-Ibraheemi5, Romaissa Boutiche6,
Naima El-Yousfi7, Mohammed Ennaji8, Ritu S Santwani9, Mohamed Zarqaoui10
1
Faculty of Medicine, Northwestern University, MD, Reproductive Biology, Assisted Reproductive Technology, Consultant, IRIFIV Fertility Center,
Administrative Deputy, Writer, ART IRIFIV Scientific Research Group (AISRG), Casablanca, Morocco
2
Newcastle University, Gynecologist and Obstetrician, Endoscopic Surgeon, Fertility Expert and Research Supervisor, Society for Art Irifiv Scientific
Research Group, Liverpool, England
3
Department of Clinical Biology, Strasbourg University Medicine, Strasbourg, Alsace-Champagne-Ardenne, FR, RB Reproductive Biology, Head,
LABOMAC Laboratory of Clinical Analysis and Assisted Reproductive Technology, IRIFIV Fertility Center, AFC Fertility Center, Executive Vice
President, ART IRIFIV Scientific Research Group (AISRG), Casablanca, Morocco
4
Gynecologist and Obstetrician, Endoscopic Surgeon and Fertility Expert, Researcher, ART IRIFIV Scientific Research Group Associated Practi-
tioner, IRIFIV Fertility Center, Casablanca, Morocco
5
Embryologist, University of Nottingham, UK, Researcher, Scientific Research Group and Member, ART IRIFIV Scientific Research Group (AISRG)
United Kingdom
6
University of Science and Technology Houari Boumediene, MA, Molecular Pathology and Biotechnology, Clinical Embryologist, IVF Laboratory in
Rotaby Fertility Center, Algeria, Researcher, Scientific Research Group, Member, ART IRIFIV Scientific Research Group (AISRG) Algeria
7
Senior Clinical Embryologist, Ibn Badis Fertility Center, Researcher, Scientific Research Group, Member, Art Irifiv Scientific Research Group
(AISRG) El Jadida, Morocco
8
Senior Clinical Embryologist, IRIFIV Fertility Center, Member, Scientific Research Group, Researcher, Scientific Research Group, Casablanca,
Morocco
9
Gynaecologist and Obstetrician, ART-Singapore, Honorary Professor, India, Member, Scientific Research Group, Researcher, Scientific Research
Group, Casablanca, Morocco
10
Strasbourg University Medicine Pole, Endoscopic Surgery Obstetrics and Gynecology Strasbourg, Alsace-Champagne-Ardenne, FR, MD. Spe-
cialist, Gynaecologist Obstetrician, General Medical Coordinator, IRIFIV Fertility Center, Head, ART IRIFIV Scientific Research Group (AISRG)
Casablanca, Morocco
Received date: 25 January 2021 ; Accepted date: 07 February 2021; published date: 14 February 2021
ABSTRACT
The primary goal is sperm retrieval. Clinical outcome of fresh testicular spermatozoa of unexpected sperm retrieval failure on the
day of egg retrieval are not common but occur more often in patients with severe oligospermia or Non-Obstructive Azoospermia
(NOA). Egg freezing is a common strategy after failed sperm collection ovarian stimulation is performed simultaneously. However,
the use of vitrification in oocytes is like these cases of male infertility are still not clear.
Objective: To investigate the oocyte outcomes arising after sperm failure Recall from patients with severe or Non-Obstructive
Oligospermia (NOA) Oocyte retrieval day.
Methods: A retrospective group study setting Society for Art Irifiv Scientific Research Group and January 2021- Patients for 203
couples with NOA (n=200) or severe oligospermia (n=3), testicular spermatozoa (n=67 cycles) or frozen sperm (n=209 cycles)
were injected into fresh or frozen-thawed oocytes via 276 Intracytoplasmic Sperm Injection (ICSI) cycles. Main Outcome Mea-
sures: Clinical pregnancy and live-birth rates (LBRs).
Results: In the 67 cycles involving the use of fresh testicular spermatozoa, no significant differences were observed between
fresh and warmed oocytes with respect to the fertilization rates.
Corresponding Author:
Zakaria M, Consultant, IRIFIV Fertility Center, Admin-
istrative Deputy and Writer, The ART IRIFIV Scientific
Research Group (AISRG), Casablanca, Morocco, E-mail: This is an open access article distributed under the terms of the Creative Com-
mons Attribution-Non Commercial-Share A like 3.0 License,which allows oth-
dr.mustafazakaria@gmail.com
ers to remix, tweak, and build upon the work non-commercially,as long as the
Steven MF, Newcastle University, Gynecologist and au-thor is credited and the new creations are licensed under the identical terms.
Obstetrician, Endoscopic Surgeon, Fertility Expert and For reprints contact: editor@jbcrs.org
Research Supervisor, Society for Art Irifiv Scientific
Research Group, Liverpool, England, E-mail: info@irifiv- Copyright: © 2021 Zakaria M. This is an open-access article distributed under
the terms of the Creative Commons Attribution License, which permits unre-
aisrg.com
stricted use, distribution, and reproduction in any medium, provided the original
DOI: 10.4103/2278-960X.1945147 author and source are credited.
© 2021 Journal of Basic and Clinical Reproductive SciencesCitation: Zakaria M et al. Clinical outcome of fresh testicular spermatozoa and failure to retrieve sperm from patients with severe or non-obstructive azoospermia of
oocyte retrieval and the in vitro fertilization/intracytoplasmic sperm injection protocol doi:10.4103/2278-960X.1945147
Conclusions: Emerging oocyte freezing is a feasible strategy for unexpected sperm management retrieval failure of oligospermia
or NOA patients on the day of oocyte retrieval. Over there it has no adverse effect on the live birth rate of frozen testicular or
failure to retrieve sperm from patients with severe or non-obstructive azoospermia of oocyte retrieval and in vitro fertilization/
intracytoplasmic sperm injection protocol.
Keywords: Clinical outcome of fresh testicular spermatozoa, IVF/ICSI protocol, Oocytes vitrification, Thawing method, Unex-
pected sperm retrieval failure, Clinical outcomes of frozen-thawed sperm.
Abbreviations: ICSI: Intracytoplasmic Sperm Injection; LBRs: Live Birth Rates; MGR: Multiple Gestation Rate; MicroTESE: Micro-
dissection Testicular Sperm Extraction; FSH: Follicle Stimulating Hormone; LH: Luteinizing Hormone; NOA: Non-Obstructive Azo-
ospermia; OPU: Oocyte Pick-Up; EG: Ethylene Glycol; LBR: The live birth rate; COH: Controlled Ovarian Hyperstimulation; MGR:
Multiple Gestation Rate; PGT-A: Preimplantation Genetic Testing for Aneuploidy
INTRODUCTION World Health Organization guidelines [5]. All patients had a
clinical work-up with physical examination, endocrine profile
In vitro fertilization/Intracytoplasmic sperm injection proto- test(Follicle Stimulating Hormone (FSH), Luteinizing Hormone
col (LH) and testosterone) and genetic analysis [6,7]. Scrotal and
Controlled ovarian stimulation, oocyte collection, and denuda- transrectal ultrasounds [8] were performed on indication. Ex-
tion were performed as previously described [1]. All patients clusion criteria for testicular biopsy were (1) previous testicu-
were administered leuprolide acetate (Lupron, Takeda Chemi- lar-sperm retrieval failure 2 times; (2) patients with a known
cal Industries, Ltd., Osaka, Japan), started during the mid-luteal abnormal karyotype and Yq deletions; (3) previous testicular
phase for down regulation. All patients subsequently received sperm from microTESE was highly pathological coupled with
recombinant follicle stimulating hormone (Gona-F; Serono, poor fertilization and poor grading of embryos. If NOA patients
Bari, Italy) for ovarian stimulation from Day 3 until the dom- were not eligible for testicular biopsy, transferring to sperm
inant follicle reached a diameter of >18 mm, followed by in- donation program was arranged for them directly. Semen was
jection of 250 μg human chorionic gonadotropin (Ovidrel, Se- originated from one specific healthy adult after matching with-
rono) 36 hours before oocyte retrieval. The retrieved oocytes out consanguinity according to country’s legislation. Frozen
were cultured in Quinn’s Advantage Fertilization Medium (Sage sperm was cleared for use after a sexually transmitted disease
BioPharma, Inc., Trumbull, CT, USA) with 15% serum protein screening 6 months after donation, in accordance with Tai-
substitute (SPS, Sage BioPharma, Inc.) in a triple gas phase of wanese law. The NOA male partners underwent microTESE on
5% CO2, 5% O2, and 90% N2. The cumulus cells were removed the oocyte retrieval day. If spermatozoa were available with
by pipetting the oocytes in modified human tubal fluid media microTESE, ICSI was performed after Oocyte Pick-Up (OPU) 3
(mHTF) containing 80 IU/mL hyaluronidase (Type 8, H-3757; hours later [9]. If surgical sperm retrieval failed, oocytes were
Sigma Chemical, St. Louis, MO, USA). Following ICSI, all embry- frozen with the vitrification method and then the couple either
os were further cultured in micro drops of Quinn’s Advantage underwent another microTESE procedure (2 or more attempts
Cleavage (SAGE) medium [2-3]. A droplet of medium with pre- depending on clinical condition) or was referred to a sperm do-
pared spermatozoa was mixed with up to 3.5% polyvinyl pyr- nation program, depending on the pathology of the testicular
rolidone (PVP, SAGE Media, USA). Metaphase-II oocytes were biopsy. For male partners with severe oligospermia, oocytes
placed in an HTF droplet and injected with one spermatozoon were also frozen if unexpected azoospermia occurred on oo-
each after gentle aspiration of the cytoplasm [4]. After ICSI, cyte retrieval day and surgical sperm retrieval was performed if
embryonic development, including the embryonic pronuclei still azoospermic. The retrospective data analysis was approved
appearance (18–20 hours) and eight-cell stage (69–70 hours), by the Institutional Review Board of Chung Shan Medical Uni-
was observed. The embryo transfer was performed on Day 3 versity, Taichung, Taiwan (CS-09054) and all patients provided
after oocyte retrieval or thawing of oocytes. Day 3 embryos written informed consent before their microTESE biopsies.
with 8 equal sized blastomeres and 20% fragmentation were MicroTESE procedure
considered good quality embryos.
The procedures were performed under local anesthesia and
METHODS started on the testicle with larger volume [10]. The scrotum
Patient selection was incised longitudinally for 3 cm on the median raphe and
the testis was then delivered through the incision. MicroTESE
This retrospective observational, single center cohort study was performed using an operative microscope (Carl Zeiss, OPMI
included 203 patients who underwent 276 intra Cytoplasmic Surgical Microscope, Germany) to expose the seminiferous
Sperm Injection (ICSI) cycles with fresh or frozen-thawed oo- tubules. The testicle was then split open bluntly and tubules
cytes for couples with NOA or severe oligospermia at the Lee were retrieved with jewelers’ forceps from different sites of
Women’s Hospital, Taiwan between March 2020 and January the two testicular sections. A fragment of testicle was washed
2021. Azoospermia was diagnosed when the absence of sperm in 1 ml mHTF [11] with 5% Serum Substitute Supplement fixed
was observed in two semen samples, in accordance with the in Bouin’s solution (1 ml) for pathology. The surgeon extracted
the tissue fragments (TESE sample) and placed it in a Petri dish.
At the end of the procedure, the albuginea incision was closed
Journal of Basic and Clinical Reproductive Sciences • Jan-July 2021 • Vol 10 • Issue 1Citation: Zakaria M et al. Clinical outcome of fresh testicular spermatozoa and failure to retrieve sperm from patients with severe or non-obstructive azoospermia of
oocyte retrieval and the in vitro fertilization/intracytoplasmic sperm injection protocol doi:10.4103/2278-960X.1945147
with a VICRYL 6/0 running suture. The embryologist opened up pregnancy was defined as the presence of an intrauterine ges-
the seminiferous tubules by mechanically dissecting the tissue tational sac with positive cardiac movement on ultrasound at
with glass cover slides before sperm search. The TESE sample 6–8 weeks [17]. Pregnancy outcome was tracked for all preg-
was then centrifuged (600 G for 10 min) and the embryologist nant women by mailed questionnaire or by phone. The Live
continued the sperm search on the more concentrated sample. Birth Rate (LBR) was defined as the proportion of IVF cycles
If sperm was not found on the day of surgery, the oocytes were reaching embryo transfer that resulted in the birth of at least
vitrified. one live-born child. Cumulative pregnancy rate was followed
up till May 2018.
Unexpected sperm retrieval failure
Unexpected sperm retrieval failure on the day of oocyte re- STATISTICAL ANALYSIS
trieval is not common but frequently happens in patients with The primary outcome measure was the live birth rate between
severe oligospermia or Non-Obstructive Azoospermia (NOA). the groups with fresh oocytes and warmed oocytes. No for-
Oocyte cryopreservation is a common strategy after failed col- mal sample size calculation was performed but all available
lection of sperm when concurrent ovarian stimulation is un- patients in our center were included in the study. Quantitative
derwent. Although concurrent ovarian stimulation for the sit- variables, representing differences between groups of patients
uation is still on debate with controversies, oocyte-freezing is in mean ± SD, and categorical variables, representing differ-
the only way if ovarian stimulation has been underwent. How- ences in distributions, were tested with the Kruskal-Wallis
ever, the use of oocyte vitrification in such male-infertility cas- one-way analysis of variance (ANOVA) and the Chi-square test,
es remains unclear. There are few reports on the outcomes of respectively. Nonnormally distributed continuous variables are
subsequent use of testicular spermatozoa via microdissection expressed as medians (interquartile range) unless otherwise
testicular sperm extraction (microTESE) or frozen sperm on stated. Continuous and categorical variables were compared
cryopreserved oocytes. Published data on oocyte vitrification/ between groups using the Kruskal Wallis test and Fisher’s exact
warming has demonstrated acceptable success rates in young, test, respectively. The statistical analysis was conducted using
highly selected populations [12]. Kushnir et al. indicated that the Statistical Program for Social Sciences (SPSS Inc., Version
fresh oocytes still represent standard of care due to higher live 15.0, Chicago, U.S.A.) and MedCalc Statistical Software version
birth rates. Moreover, data on the feasibility of using frozen 16.8 (MedCalc Software, Ostend, Belgium; 2016). All p-values
oocytes for male-factor infertility is lacking. Compared with were two-sided, and values less than 0.05 were considered sta-
fresh oocytes, the main concern is the decrease in the success tistically significant.
rate when using frozen eggs for severe male infertility. There-
fore, we conducted a retrospective study to examine the clini- RESULTS
cal outcomes of emergent oocyte cryopreservation after failed Clinical outcome of fresh testicular spermatozoa
sperm retrieval from severely oligospermic or NOA patients on
oocyte retrieval day. During the study period, 276 fresh ICSI cycles from 203 couples
were included in the retrospective study. A total of 67 cycles
Oocytes vitrification or thawing method used fresh testicular spermatozoa to inseminate the fresh or
Oocyte vitrification occurred 1-2 hours after cumulus removal. warmed oocytes. 11 cycles (3 cycles from severely oligospermic
All the oocytes were vitrified and warmed using the Cryotop patients and 8 cycles from NOA patients) used frozen eggs be-
(Kitazato Co., Fujinomiya, Japan) and Cryotech Vitrification cause of previous sperm retrieval failure with unexpected egg
Method (Cryotec, Cryotech, Tokyo, Japan) [13-16]. Briefly, oo- freezing at that time. Table 1 illustrates the baseline character-
cytes were equilibrated at room temperature in the solution istics and clinical outcome of using fresh testicular spermato-
supplied with the kit containing Ethylene Glycol (EG) and Di- zoa ICSI cycles. The sperm retrieval rate of micro TESE for NOA
methylsulphoxide (Me2SO) for 15 minutes. Then the oocytes patients was 45.7%. The post-thawing survival rate of warmed
were moved to a vitrification solution containing a double eggs was 87.2% and 3 cases were cancelled in the warmed egg
concentration of EG+Me2SO, and sucrose or trehalose for 60- group due to no embryo to transfer (cancellation rate: 27%).
90 s. Oocytes were then loaded on the top of the film strip No significant differences between the groups of fresh oo-
supplied with the kit, and the sample was quickly immersed cytes and warmed oocytes were observed in fertilization rates
into liquid nitrogen for storage. At warming, the strip was im- (69.2% vs. 74.1%; p=0.27), number of Day 3 embryos (8.6 ± 4.4
mersed directly into the kit warming solution at 37˚C for 1 min vs. 6.4 ± 3.4; p=0.08), number of good-quality Day-3 embryos
and then oocytes were incubated once for 3 min and twice for (4.5 ± 3.9 vs. 4.7 ± 3.0; p=0.45), implantation rates (29.1%
5 min in the kits’ diluent and washing solutions, respectively. vs. 17.8%; p=0.21), clinical pregnancy rates (36.4% vs. 26.8.0%;
After warming, oocytes were cultured in HTF medium for 2–3 p=0.81), or live birth rates (36.4% vs. 14.3%; p=0.46). Obstetric
hours and then used for ICSI. Embryo transfer was performed outcomes indicated 4 pregnancies with 6 healthy infants born
on day 3 after oocyte retrieval or thawing oocytes. In fresh (4 singletons and 1 set of twins) in the warmed-oocyte group
oocytes group, luteal phase support was started after egg-re- and 8 pregnancies with 10 healthy infants born (6 singletons
trieval with Crinone (1.125 g, 8% gel; vaginal suppositories, and 2 sets of twins) in the fresh-oocyte group. All 15 infants
Merck Serono, UK) application daily and oral dydrogesterone had normal karyotypes. No differences in perinatal outcomes
(10 mg; Duphaston, Abbott Biologicals B.V., the Netherlands) were noted between the two groups.
three times a day for 14 days. In thawing oocytes group, pa-
tients underwent an artificial cycle for endometrial preparation
with hormone replacement as previously published. Clinical
Journal of Basic and Clinical Reproductive Sciences • Jan-July 2021 • Vol 10 • Issue 1Citation: Zakaria M et al. Clinical outcome of fresh testicular spermatozoa and failure to retrieve sperm from patients with severe or non-obstructive azoospermia of
oocyte retrieval and the in vitro fertilization/intracytoplasmic sperm injection protocol doi:10.4103/2278-960X.1945147
Table 1: Characteristics and outcomes of ICSI cycles using fresh because spermatogenesis is limited in NOA patients, surgical
Table 1: Characteristics and outcomes of ICSI cycles using fresh testicular spermatozoa in fresh oocytes or
testicular
warmed oocytes.spermatozoa in fresh oocytes or warmed oocytes. sperm retrieval rates are between 36%-64% [26]. Although sever-
al micro-surgical methods can improve surgical sperm retrieval
Warmed Oocytes Fresh Oocytes p value
No. of cycles 11 56 rates [27–30], failure of spermatozoa retrieval is still possible.
No. of patients 10 50 Emergent oocyte cryopreservation is the alternative option if
Age of male partners (y) 38.9 ± 3.9 38.9 ± 6.7 0.92
Serum FSH (mIU/ml) 15.9 (10.8-2.1) 18.6 (15.9-26.4) 0.75
concurrent ovarian stimulation is performed. However, few
Serum LH (Miu/ML) 5.1(3.6-6.6) 7.5 (6.1-8.9) 0.81 studies have reported the efficacy of emergent oocyte cryo-
Serum testoterone (ng/ml) 1.28 (0.65-1.91) 1.7 (0.61-2.81) 0.87 preservation after failed retrieval of spermatozoa. Our study
Age of female partners (y) 33.8 ± 3.1 35.6 ± 3.9 0.16
Day 3 serum FSH (Miu/ml) 5.53 ± 2.3 6.2 ± 2.3 0.38
reported similar outcomes between fresh and frozen oocytes
Duration of infertility (y) 4.6 ± 3.1 3.57 ± 3.3 0.37 when performing ICSI using surgically retrieved fresh sperma-
No. of oocytes retrieved 16.4 ± 10 12.1 ± 7.5 0.11 tozoa. Frozen oocytes were also competent to be fertilized
No. of mature oocytes retrieved 13 ± 5.4 10.2 ± 5.4 0.24
Fertilization rate, % (n) 69.2% (90/130) 74.1% (403/544) 0.27
with testicular spermatozoa and progress to healthy embry-
No. of good quality day 3 embryos 8.6 ± 4.4 6.4 ± 3.4 0.08 os. Using the surgically retrieved fresh or frozen spermatozoa
No. of embryos transferred 4.5 ± 3.9 4.7 ± 3.0 0.45 for ICSI in NOA patients is still controversial, with contrasting
Implantation rate, % (n) 2.2 ± 1.5 1.8 ± 1.6 0.48
Clinical pregnancy rate, % (n) 29.1% (7/24) 17.8% (18/101) 0.21
results. The advantages of frozen testicular spermatozoa are
Live birth rate, % (n) 36.4% (4/11) 26.8% (15/56) 0.81 the independent scheduling of sperm and oocyte retrieval,
Abortion rate, % (n) 36.4% (4/11) 14.3% (8/56) 0.46 avoidance of unnecessary ovarian stimulation of the female
Multiple gestation, % (n) 0% (0/4) 30% (5/15) 0.18
Gestation age (weeks) 25% (1/4) 25% (2/8) 1
partner (if no sperm is retrieved) and concomitant lessening
Birth weight (gm) 37.7 ± 0.5 37.7 ± 1.2 1 of stress to the couple. Although most relevant studies [31–36]
3037 ± 315 2881 ± 373 0.49 suggest that fertilization rates using cryopreserved-thawed
Note: Values are % (n) or mean ± SD or medians (interquartile range); PCitation: Zakaria M et al. Clinical outcome of fresh testicular spermatozoa and failure to retrieve sperm from patients with severe or non-obstructive azoospermia of
oocyte retrieval and the in vitro fertilization/intracytoplasmic sperm injection protocol doi:10.4103/2278-960X.1945147
ful live birth after emergency oocyte cryopreservation when 8. Sakamoto H, Yajima T, Nagata M, et al. Relationship
sperm extraction failed has been reported [48,49]. However, between testicular size by ultrasonography and tes-
the importance of emergent oocyte cryopreservation has not ticular function: measurement of testicular length,
been emphasized for this situation. According to our results, width, and depth in patients with infertility. Int J Urol.
emergent oocyte cryopreservation is an effective strategy to 2008;15(6):529–33.
allow for arranging further biopsies and medical treatment
or to seek an eligible. Segmentation of assisted reproductive 9. Palermo G, Joris H, Devroey P, et al. Pregnancies after in-
technology into ovarian stimulation and sperm retrieval from tracytoplasmic injection of single spermatozoon into an
surgery could have the potential to improve and extend the oocyte. Lancet. 1992; 340(8810):17–8.
current assisted reproductive technologies. The present study 10. Schlegel PN. Testicular sperm extraction: microdissection
has several limitations. First, the study had a small sample size. improves sperm yield with minimal tissue excision. Hum
Thus, a larger sample size is needed to confirm the results of Reprod. 1999; 14(1):131–5.
this study. Second, the retrospective study model has a risk of
11. Shih YF, Tzeng SL, Chen WJ, et al. Effects of Synthetic
selection bias because the groups originally were assigned for
Serum Supplementation in Sperm Preparation Media on
non-random reasons. The male patients who were eligible to
Sperm Capacitation and Function Test Results. Oxid Med
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Cell Longev. 2016; 1027158.
cerns on LBR and costs. Therefore, the results of the study may
only represent parts of the groups not all due to these limita- 12. Chen HH, Huang CC, Cheng EH, et al. Optimal timing
tions. A randomized controlled trial could improve the quality of blastocyst vitrificationa fter trophectoderm biopsy
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13. Lee CI, Lee TH, Huang CC, et al. Detection of early cleav-
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